Methods for measuring fat digestibility and uses thereof

ABSTRACT

The present invention provides methods for continuous measurement of triglyceride digestibility during a meal. A stable isotope labeled triglyceride and a free fatty acid tracer are added to the meal. The ratio between a ratio of isotope labeled fatty acid produced after digestion to free fatty acid tracer represents the percentage of digestion of triglycerides by lipase from the pancreas.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional under 35 U.S.C. § 120 of pendingnon-provisional U.S. Ser. No. 14/031,921, filed Sep. 19, 2013, whichclaims benefit of priority under 35 U.S.C. § 119(e) of provisionalapplication U.S. Ser. No. 61/703,582, filed Sep. 20, 2012, the entiretyof which is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention is in the field of nutrition. More specifically,the present invention is directed to methods of measuring protein and/orfat digestibility and uses thereof.

Description of the Related Art

Malabsorption of nutrients due to exocrine pancreatic insufficiency is amain factor contributing to weight loss or poor weight gain in patientswith cystic fibrosis (CF) leading to deficiencies of essentialnutrients. Although it is well established that fat digestibility isimpaired in cystic fibrosis, the prior art methods to measure proteindigestibility are limited and often not accurate. As impaired proteindigestibility leads to an impaired anabolic response to a meal as wellas to high loads of undigested proteins in the colon, which inducediarrhea and the production of harmful toxins by gut bacteria, accuratequantification of protein digestibility is of high clinical importancein cystic fibrosis.

Many decades ago, a diminished retention of dietary proteins in cysticfibrosis was demonstrated with nitrogen balance tests and stoolanalyses, methods with limited accuracy that are too laborious and timeconsuming for routine diagnostic use. In 1952, a simpler and accuratemethod was developed using oral ingestion of ¹³¹I-labeled protein. ¹³Cintrinsically labeled milk or egg proteins has been used to assessprotein digestibility by measuring the enrichment kinetics of labeledCO₂ in the breath in response to meal intake. A limitation of thismethod is the difficulty to obtain large amounts of proteins withsufficiently high amino acid enrichment levels and with adequatelabeling patterns, making production of these labeled proteinscumbersome and expensive. Furthermore, the accuracy of using¹³C-intrinsically labeled milk or egg protein, or even uniformly¹³C-labeled algal protein and measuring ¹³CO₂ production might belimited in cystic fibrosis as these patients are characterized bychronic and acute episodes of lung inflammation which contribute to CO₂production in the breath and changes in the CO₂ pool size.

Reduced digestion capacity of nutrients due to exocrine pancreaticinsufficiency (EPI) is an important disease-related factor contributingto weight loss. Severe exocrine pancreatic insufficiency impairs thedigestion of fat, carbohydrate and protein leading to nutrientdeficiencies. Current dietary recommendations comprise a high energy(>120% of recommended daily allowances), high fat and high protein diet(40% and 15%, respectively). Since high dietary fat is the main energysource for patients with cystic fibrosis, fat absorption is currentlythe marker of effective nutrient digestion in cystic fibrosis patients.In addition, providing an appropriate dose and good timing of PancreaticEnzyme Replacement Therapy (PERT) is of crucial importance to increaselipid digestibility and systemic lipid availability in CF.

In refractory patients who do not respond to Pancreatic EnzymeReplacement Therapy, fecal energy content and fecal fat balance study isthe non-invasive gold standard method to assess pancreatic exocrinefunction by measuring the consequence of reduced fat digestion andabsorption. This technique is based on a measurement of triglycerides infeces during a three day collection period and careful determination offat intake to be able to calculate the fat balance. In daily practice,the appropriate dose of Pancreatic Enzyme Replacement Therapy isdetermined on clinical ground (symptoms and weight gain) alone, becausethe fecal fat balance test is unpleasant, unpractical, and cumbersome.

No information is available regarding the acute effect of PancreaticEnzyme Replacement Therapy on lipid digestion in CF, which is ofparticular importance in patients who receive continuous night timegastro-intestinal tube feeding. When pancreatic enzymes are given at thebeginning of enteral feeding, the duration of enzyme activity, absolutenecessity and timing of a second dose, and appropriate dose to improvefat digestion are unknown. Because the current standard techniquemeasures over a 72-hour period, this technique represents a long termfat digestion and absorption capacity and cannot be used to measureacute changes in fat digestibility. Thus, there is no establishedtechnique available to measure acute fat digestibility in CF.

Techniques that use carbon-labeled radioactive or stable isotope fattyacids have been described, measuring the labeled CO₂ production afteringestion of the labeled fatty acids. These methods have been used inpatients with CF to detect fat malabsorption and to monitor the efficacyof Pancreatic Enzyme Replacement Therapy. However these methods haveseveral disadvantages (e.g., the results are a combination of theprocess of fat digestion and absorption, and are related to metabolismand oxidation of fatty acids to CO₂ in the body) and therefore are notused anymore to calculate fat uptake in CF.

The prior art is deficient in techniques for the measurement of proteinand/or fat digestibility. The present invention fulfills thislongstanding need and desire in the art.

SUMMARY OF THE INVENTION

The present invention is directed to a method for determining reducedprotein digestibility in an individual in need of such determination,comprising the step of: administering a meal comprising an isotopelabeled protein and a free amino acid tracer to said individual and to acontrol subject; and measuring the amount of isotope labeled amino acidformed after digestion of said isotope labeled protein and measuring theamount of free amino acid tracer in a biological sample of saidindividual and said control subject to determine a ratio of isotopelabeled amino acid to free amino acid tracer for said individual and forsaid control subject; wherein a lower ratio of labeled amino acid tofree amino acid tracer in said individual compared to the ratio oflabeled amino acid to free amino acid tracer from said control subjectindicates that said individual has a reduced capacity to digest protein.

The present invention is directed further to a method for obtainingprotein digestibility profile in an individual, comprising the step of:administering a meal comprising an isotope labeled protein and a freeamino acid tracer to said individual; measuring the amount of isotopelabeled amino acid formed after digestion of said isotope labeledprotein in a biological sample of said individual and measuring theamount of free amino acid tracer in the biological sample to determine aratio of isotope labeled amino acid to free amino acid tracer; andrepeating said measuring step one or more times to obtain a proteindigestibility profile for said individual.

The present invention is directed further still to a kit for determiningreduced protein digestibility, comprising: an isotope labeled protein; afree amino acid tracer; and instructions for determining proteindigestibility.

In another preferred embodiment, the present invention is directed to amethod for determining reduced fat digestibility in an individual inneed of such determination, comprising the step of: administering a mealcomprising a stable isotope labeled triglyceride and a free fatty acidtracer to said individual and a control subject; and measuring theamount of isotope labeled fatty acid formed after digestion of saidisotope labeled triglyceride and measuring the amount of free fatty acidtracer in a biological sample of said individual and said controlsubject to determine a ratio of isotope labeled fatty acid to free fattyacid tracer for said individual and said control subject; wherein alower ratio of labeled fatty acid to free fatty acid tracer in saidindividual compared to the ratio of labeled fatty acid to free fattyacid tracer from said control subject indicates that said individual hasa reduced capacity to digest fat.

The present invention is directed further to a method for obtaining fatdigestibility profile in an individual, comprising the step of:administering a meal comprising a stable isotope labeled triglycerideand a free fatty acid tracer to said individual; measuring the amount ofisotope labeled fatty acid formed after digestion of said isotopelabeled triglyceride and measuring the amount of free fatty acid tracerin a biological sample of said individual to determine a ratio ofisotope labeled fatty acid to free fatty acid tracer; and repeating saidmeasuring step one or more times to provide a fat digestibility profilefor said individual.

The present invention is directed further still to a kit for determiningreduced fat digestibility, comprising: an isotope labeled triglyceride;a free fatty acid tracer; and instructions for determining fatdigestibility.

Other and further aspects, features, and advantages of the presentinvention will be apparent from the following description of thepresently preferred embodiments of the invention. These embodiments aregiven for the purpose of disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

So that the matter in which the above-recited features, advantages andobjects of the invention, as well as others which will become clear, areattained and can be understood in detail, more particular descriptionsand certain embodiments of the invention briefly summarized above areillustrated in the appended drawings. These drawings form a part of thespecification. It is to be noted, however, that the appended drawingsillustrate preferred embodiments of the invention and therefore are notto be considered limiting in their scope.

FIGS. 1A-1B show the principle to measure protein digestibility (FIG.1A) and fat digestibility (FIG. 1B).

FIG. 2 outlines an overview of the study design for administeringnutrition, stable isotopes and pancreatic enzyme intake.

FIGS. 3A-3B show the response in protein digestibility during feedingand after intake of pancreatic enzymes (only in CF) in the whole CFgroup (dashed line) and healthy adult group (solid line) (FIG. 3A), andin the healthy group (solid line) and after stratification of the wholeCF group into adults (dashed line) and children (dotted line) (FIG. 3B).

FIGS. 4A-4D shows postabsorptive whole body rate of appearance (WbRa,FIGS. 4A, 4C) and production rate (FIGS. 4B, 4D) of citrulline in thehealthy adult group (solid bar) and after stratification of the whole CFgroup into adults (open bar) and children (cross striped bar), and afterstratification of the CF group into nutritional failure (NF, verticalstriped bar) and no nutritional failure (no NF, horizontal striped bar).Mean values±SE are shown. Significance of difference as compared to thehealthy group (*: P<0.05, **: P<0.01) and as compared to the CF groupwith no nutritional failure (^(#): P<0.05).

FIGS. 5A-5B show the tracer tracee ratio response of Palmitate mass 2(dashed line) and mass 1 (dotted line) in the CF adult (FIG. 5A) and CFchildren (FIG. 5B).

FIGS. 6A-6B show the response in fat digestion rate in CF group (FIG.6A), compared to the fat digestion rate in the healthy control group(dashed line; set at 100%) and after stratification of the whole CFgroup into adults (dashed line) and children (dotted line), duringfeeding and after intake of pancreatic enzymes (FIG. 6B; only in CF).

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common scientific technical terms maybe found, for example, in Mcgraw-hill Dictionary of Scientific &Technical Terms published by Mcgraw-hill Healthcare Management Group;Benjamin Lewin, Genes VIII, published by Oxford University Press;Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, publishedby Blackwell Publishers; and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by Wiley,John & Sons, Inc; and other similar technical references.

As used herein the specification, “a” or “an” may mean one or more. Asused herein in the claim(s), when used in conjunction with the word“comprising”, the words “a” or “an” may mean one or more than one. Asused herein “another” may mean at least a second or more. Furthermore,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular.

As used herein, the term “or” in the claims refers to “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or”.

As used herein, the term “about” refers to a numeric value, including,for example, whole numbers, fractions, and percentages, whether or notexplicitly indicated. The term “about” generally refers to a range ofnumerical values (e.g., +/−5-10% of the recited value) that one ofordinary skill in the art would consider equivalent to the recited value(e.g., having the same function or result). In some instances, the term“about” may include numerical values that are rounded to the nearestsignificant figure.

As used herein, the terms “subject” or “individual” refers to a mammal,preferably a human, who is a recipient of any therapeutic agent or othermolecules described herein.

As used herein, the term “enteral administration” refers to route ofdrug administration that involves absorption of the drug through thegastrointestinal tract.

In one embodiment of the present invention, there is provided a methodfor determining reduced protein digestibility in an individual in needof such determination, comprising the step of: administering a mealcomprising an isotope labeled protein and a free amino acid tracer tosaid individual and to a control subject; and measuring the amount ofisotope labeled amino acid formed after digestion of said isotopelabeled protein and measuring the amount of free amino acid tracer in abiological sample of said individual and said control subject todetermine a ratio of isotope labeled amino acid to free amino acidtracer for said individual and for said control subject; wherein a lowerratio of labeled amino acid to free amino acid tracer in said individualcompared to the ratio of labeled amino acid to free amino acid tracerfrom said control subject indicates that said individual has a reducedcapacity to digest protein. In a non limiting example, the subject hascystic fibrosis. In aspects of this embodiment, representative examplesof an isotope labeled protein include but are not limited to ¹⁵N-labeledspirulina protein, ¹⁵N-labeled milk protein, ¹⁵N-labeled soy protein, or¹⁵N-labeled wheat protein wherein said isotope labeled protein comprisesan isotope-labeled amino acid. For example, the isotope-labeled aminoacid may be ¹⁵N-labeled amino acid, ¹³C-labeled amino acid or ²H labeledamino acid. Representative examples of labeled amino acid include butnot limited to ²H-phenylalanine, ²H-lysine, ²H-leucine,¹⁵N-phenylalanine, ¹⁵N-lysine, ¹⁵N-leucine, ¹³C-phenylalanine,¹³C-lysine, or ¹³C-leucine. In aspects of this embodiment, the freeamino acid tracer is ²H-labeled amino acid, ¹⁵N-labeled amino acid or¹³C-labeled amino acid. For example, the free amino acid tracer may be²H-phenylalanine, ²H-lysine, ²H-leucine, ¹⁵N-phenylalanine, ¹⁵N-lysine,¹⁵N-leucine, ¹³C-phenylalanine, ¹³C-lysine, or ¹³C-leucine. In allembodiments, the biological sample is blood or plasma.

Provided herein is kit for determining reduced protein digestibility,comprising: an isotope labeled protein; a free amino acid tracer; andinstructions for determining protein digestibility.

In another embodiment of the invention, there is provide a method forobtaining protein digestibility profile in an individual, comprising thestep of: administering a meal comprising an isotope labeled protein anda free amino acid tracer to said individual; measuring the amount ofisotope labeled amino acid formed after digestion of said isotopelabeled protein in a biological sample of said individual and measuringthe amount of free amino acid tracer in the biological sample todetermine a ratio of isotope labeled amino acid to free amino acidtracer; and repeating said measuring step one or more times to obtain aprotein digestibility profile for said individual. In the aspects ofthis embodiment, said isotope labeled protein is ¹⁵N-labeled spirulinaprotein, ¹⁵N-labeled milk protein, ¹⁵N-labeled soy protein, or¹⁵N-labeled wheat protein. The free amino acid tracer is²H-phenylalanine, ²H-lysine, ²H-leucine, ¹⁵N-phenylalanine, ¹⁵N-lysine,¹⁵N-leucine, ¹³C-phenylalanine, ¹³C-lysine, or ¹³C-leucine.

In yet another preferred embodiment of the invention, there is a methodfor determining reduced fat digestibility in an individual in need ofsuch determination, comprising the step of: administering a mealcomprising a stable isotope labeled triglyceride and a free fatty acidtracer to said individual and a control subject; and measuring theamount of isotope labeled fatty acid formed after digestion of saidisotope labeled triglyceride and measuring the amount of free fatty acidtracer in a biological sample of said individual and said controlsubject to determine a ratio of isotope labeled fatty acid to free fattyacid tracer for said individual and said control subject; wherein alower ratio of labeled fatty acid to free fatty acid tracer in saidindividual compared to the ratio of labeled fatty acid to free fattyacid tracer from said control subject indicates that said individual hasa reduced capacity to digest fat. In a non-limiting example, the subjecthas cystic fibrosis. In aspects of this embodiment, representativeexamples of stable isotope labeled triglyceride include but not limitedto [1,1,1-¹³C₃]tripalmitin, [1,1,1-¹³C₃]triolein, [U—¹³C₁₆]tripalmitin,or [U—¹³C₁₈]triolein. The isotope labeled fatty acid formed afterdigestion of said isotope labeled triglyceride may be [1-¹³C]palmiticacid, [1-¹³C]oleic acid, [U—¹³C¹⁶]palmitic acid, or [U—¹³C₁₈]oleic acid.Representative examples of free fatty acid tracer include but notlimited to [2,2-²H₂]palmitic acid, [2,2-²H₂]oleic acid,(7,7,8,8-D₄)palmitic acid, (9,10-D₄)oleic acid. In all embodiments, thebiological sample is blood or plasma.

Provided herein is a kit for determining reduced fat digestibility,comprising: an isotope labeled triglyceride; a free fatty acid tracer;and instructions for determining fat digestibility.

In yet another embodiment of the invention, there is a method forobtaining fat digestibility profile in an individual, comprising thestep of: administering a meal comprising a stable isotope labeledtriglyceride and a free fatty acid tracer to said individual; measuringthe amount of isotope labeled fatty acid formed after digestion of saidisotope labeled triglyceride and measuring the amount of free fatty acidtracer in a biological sample of said individual to determine a ratio ofisotope labeled fatty acid to free fatty acid tracer; and repeating saidmeasuring step one or more times to provide a fat digestibility profilefor said individual. In the aspects of this embodiment, said stableisotope labeled triglyceride is [1,1,1-¹³C₃]tripalmitin,[1,1,1-¹³C₃]triolein, [U—¹³C₁₆]tripalmitin, or [U—¹³C₁₈]triolein. saidfree fatty acid tracer is [2,2-²H₂]palmitic acid, [2,2-²H₂]oleic acid,(7,7,8,8-D₄)palmitic acid, or (9,10-D₄)oleic acid.

The following examples are given for the purpose of illustrating variousembodiments of the invention and are not meant to limit the presentinvention in any fashion. One skilled in the art will appreciate readilythat the present invention is well adapted to carry out the objects andobtain the ends and advantages mentioned, as well as those objects, endsand advantages inherent herein. Changes therein and other uses which areencompassed within the spirit of the invention as defined by the scopeof the claims will occur to those skilled in the art.

Example 1 Protein Digestion Study Materials and Methods

Subjects

Nineteen (19) subjects with cystic fibrosis (CF) were studied of whichten were pediatric subjects, age of 10 to 18 years, and admitted toArkansas Children's Hospital for pulmonary exacerbation. Nine of the CFpatients were adults, age of 18 to 35 years at the time of enrollment.Three of the adult patients were admitted to the University of Arkansasfor Medical Sciences for pulmonary exacerbation, and 6 were outpatients.All CF subjects had a diagnosis of CF based on universal diagnosticcriteria, were clinically stable at enrollment, and were pancreaticinsufficient. The CF inpatients were enrolled at the end of theirhospital stay with improvement in lung function (FEV₁) at the time ofenrollment back to baseline values (determined as FEV₁ in past 12months). Exclusion criteria included established diagnosis of diabetesmellitus, unstable metabolic diseases, and chronic respiratory failurewith cor pulmonale. In addition, 8 healthy subjects were recruited inthe local community and studied as age-matched control subjects to theadult CF patients. Written informed consent was obtained and the studywas approved by the Institutional Review Board of the University ofArkansas for Medical Sciences. This trial is registered atClinicalTrials.gov under as NCT01494909.

Study Design

The study day was performed in the patient's hospital room during thelast days of antibiotic treatment for a CF exacerbation at ArkansasChildren's Hospital (CF children, n=10) or University of Arkansas forMedical Sciences (CF adults, n=3). Moreover, 6 adults with CF and 8healthy adults were studied at the outpatient clinical research centerat University of Arkansas for Medical Sciences. The study day started inthe early morning after overnight fasting and lasted for 8 hours. Ifpresent, a central-venous port or peripheral line already in place forclinical care was used; otherwise, a catheter was placed in asuperficial vein of the lower arm. This line was used for backgroundblood sampling, the bolus infusion of the stable isotope of citrulline(L-[5-¹³C-5,5-²H₂]-Cit) (Sigma-Aldrich; St. Louis, Mo.) as well as forsubsequent blood sampling. Two hours after the IV citrulline bolus, eachsubject ingested orally or received enterally (when feeding tube waspresent in CF) a commercially available nutritional supplement accordingto a sip feeding protocol (each 20 min) during 6 hours. The oralisotopes of ¹⁵N-spirulina and L-[ring-²H₅]Phenylalanine (CambridgeIsotopic Laboratories, Andover, Mass.) were added to the nutritionalsupplement. After 2 hours of sip feeding, one serving of pancreaticenzymes (Creon®, 4000 u lipase/g fat intake) (Abbott; Abbott Park, Ill.)was ingested by the CF subjects. No pancreatic enzymes were taken by thehealthy adults. Arterialized-venous blood samples were taken throughoutthe study for analysis of concentrations and tracer-tracee ratios (TTR)of amino acids. An overview of study design is outlined in FIG. 2.

Composition of the Nutritional Supplement and Pancreatic Enzyme Dose

All subjects received a commercially available nutritional supplement,Ensure Plus® (Abbott Nutrition; Abbott Park, Ill.), which is often usedin CF care. The subjects ingested the nutritional supplement orallyaccording to a sip feeding protocol (every 20 min) or enterally when agastrostomy tube was present (CF children, n=3). The dosage of thenutritional supplement for each subject was based on ⅓^(rd) of theirtotal daily protein needs, estimated using Dietary Reference Intakes(DRI) (for composition see Table 1). The stable isotopes ¹⁵N-spirulinaprotein (63 mg/kg BW Spirulina protein that contains about 1.7 mgL-[¹⁵N]phenylalanine (PHE)) and L-[ring-²H₅]PHE (3.14 mg/kg BW) wasadded to the nutritional supplement (3.6 g/kg BW Ensure Plus®) tomeasure protein digestion rate (=ratio [¹⁵N]PHE to [²H₅]PHE in plasma)(for an outline of principle physiology, see FIG. 1). The nutritionalsupplement was mixed with the oral stable isotopes and divided intoequal portions and stored in the refrigerator before use. A sample ofthe supplement was stored for composition and PHE isotope ratioanalysis.

Pancreatic enzyme (Creon®) intake in CF took place at 2 h into feeding,and the dose was based on the fat content of the meal (4000 u lipase/gfat) (Table 1). The pancreatic enzymes for the children and adults withCF were obtained from the Arkansas Children Hospital and University ofArkansas for Medical Sciences pharmacy, respectively.

TABLE 1 Nutrition, pancreatic enzyme and oral isotope dose used inchildren and adults with Cystic Fibrosis and healthy adults Healthyadults CF adults CF children Ensure plus ml 260.1 ± 36.9 224.3 ± 37.3170.8 ± 39.4 Creon 12000 U 0  3.56 ± 0.53  2.5 ± 0.53 Creon 6000 U 0 0.11 ± 0.33  0.50 ± 0.53 ¹⁵N-Spirulina mg 4572.8 ± 648.5 3943.3 ± 656.23003.0 ± 693.1 ²H₅- mg 228.6 ± 32.5 197.2 ± 32.8 150.1 ± 34.6Phenylalanine Values are means ± SEMAnthropometric Data and Body Composition

In the early morning of the study day, body weight and height weremeasured in all subjects by a digital beam scale and stadiometer,respectively. BMI was calculated by dividing body weight by squaredheight and expressed in kg/m² for the adults. Height, weight, and BMIpercentiles of the CF children were calculated in accordance with the CFconsensus report (1). Whole-body fat mass (FM) and fat-free mass (FFM)were obtained by dual-energy X-ray absorptiometry (DXA) (Hologic QDR4500/Version 12.7.3.1; Bedford, Mass.) when the subjects were in supineposition. The DXA procedure was conducted in the outpatient researchsetting on the study day, or obtained during hospital stay in case ofhospital admission.

The anthropometric and body composition data were standardized forheight to obtain BMI, fat-free mass index (FFMI), and fat mass index(FMI). FFMI and FMI were expressed as percentage of published referencedata Nutritional failure was defined as FFMI<5th percentile inaccordance to previous studies in CF and/or BMI<10th percentile (age≤20years) or BMI<18 kg/m2 (age 21 years and older) using the CysticFibrosis Foundation selected BMI cut-off point.

Lung Function

Forced expiratory volume in 1 second (FEV₁) and forced vital capacity(FVC) were measured by spirometry (nSpire Health; Longmont, Colo.) inall CF participants and reference equations were used to calculate FEV₁and FVC % predicted values.

Biochemical Analysis

Blood samples were put in Li-heparinized tubes, immediately put on iceand instantly frozen and stored at −80° C. until further analyses.Samples of the nutritional supplement were hydrolyzed in 6N HCl solutionfor 24 h at 110° C. All samples obtained were analyzed in a batch.Analysis for enrichment and concentrations was done by LC-ESI-MS (QTrap5500MS) (AB Sciex; Foster City, Calif.) with ExpressHT Ultra LC(Eksigent AB Sciex; Foster City, Calif.) after derivatization with9-fluorenylmethoxycarbonyl (Fmoc). FMoc-PHE and Fmoc-CIT were fragmentedto obtain specific and high sensitivity fragments.

Calculations for Protein Digestibility and Whole Body CitrullineProduction

Stable isotope of ¹⁵N-spirulina protein and ²H₅-phenylalanine was addedto the nutritional supplement (FIG. 1A) in which ²H₅-phenylalanine doesnot need digestion before absorption (digestibility=100%). Proteindigestibility is calculated by measuring the ratio [¹⁵N]PHE to [²H₅]PHEin plasma and the nutrition.

Protein digestibility was calculated by dividing the plasma [¹⁵N]PHE to[²H₅]PHE ratio by the [¹⁵N]PHE to [²H₅]PHE ratio in the nutritionalsupplement. Protein digestion rate during feeding was calculated as theaverage value at 200 min to 240 min into the study (=80-120 min afterstart of sip feeding). Highest plateau value of protein digestion rateafter intake of pancreatic enzymes was calculated as the average valueat 340 min to 400 min into the study (=100-160 min after intake ofpancreatic enzymes in CF).

To assess whole-body citrulline production in the postabsorptive statewith a pulse of citrulline isotope, the program SAAM II (Version 2.2)(The Epsilon Group; Charlottesville, Va.) was used to calculate the kvalues and pool sizes in a two compartmental model. The k values wereconverted to whole-body rate of appearance (WbRa) or intracellularproduction as described (2). The compartmental modeling estimates theparameters to calculate WbRa and intracellular production rates inrelation to the actual intracellular precursor pool enrichment. Theserates are higher than when estimated with primed-constant infusionproduction, and in that case, the precursor pool enrichment in arterialplasma is used as a proxy of intracellular enrichment, which is higherthan the intracellular enrichment as the production takes placeintracellularly.

Statistical Analysis

Results are expressed as mean±standard error (SE). Data failing thenormality or equal variance test were log-transformed where appropriate.One-way ANOVA was used to determine differences between the children andadults with CF and the healthy subjects, and Newman-Keuls was used aspost hoc analysis. Unpaired Student's t test was used to determinedifferences in clinical changes between the children and adults with CFgroup, and in the CF group with and without nutritional failure. Thelevel of significance was set at p<0.05. The statistical package withinGraphpad Prism (Version 6.01) and SPSS (Version 20) was used for dataanalysis.

Example 2 Protein Digestion Study Results

The group consisted of 10 children with CF (age: 14.9±0.2 y), 9 adultswith CF (age: 28.6±0.8 y), and 8 healthy adults (age: 29.2±1.2 y) (Table2), Age of the CF adults was not different from the healthy adults. Thehomozygous DF 508 gene was present in 90% of the children and in 25% ofthe adults with CF.

The patients with CF were characterized by mild to severe airflowobstruction. Mean FEV₁ tended to be lower (P=0.08) and FVC wassignificantly (P<0.05) lower in the CF adults than in the CF children.The CF adult group was characterized by reduced values for FFMI (aspercentage of control values: P<0.05), but no difference in FFM wasfound between the children and adults with CF. Mean BMI (in kg/m²) wasnot different between the adults with CF and the healthy subjects. Themean BMI of the CF children was at the 46 percentile indicating lowerthan the CF recommended BMI of 50%. Three adults (33%) and 3 childrenwith CF (30%) were characterized by nutritional failure.

TABLE 2 General characteristics of the children and adults with CysticFibrosis and healthy adults Healthy CF CF adults adults P children PGender m/f 4/4 5/4 6/4 Age Y 29.2 ± 1.2 28.6 ± 0.8 14.9 ± 0.2 <0.001^(a)BMI Kg/m² 23.5 ± 0.4 21.6 ± 0.5 46.4 ± 3.0 Percentile FFMI % norm 102.3± 1.6  90.7 ± 1.5 0.05 92.8 ± 1.1 FMI % norm 113.0 ± 17.3  99.4 ± 18.1 96.8 ± 10.0 Nutritional y/n 0/8 3/6 3/7 failure FEV₁ % pred 63.9 ± 3.381.6 ± 1.3 0.08^(a) FVC % pred 76.6 ± 3.6 98.0 ± 1.6 0.04^(a) CFDF508/DF508, DF508/G DF508/DF50 genotype 542x, unknown: n = 2, 8: n = 9;DF508/2184delA, DF508/1717- DF508/G1244: n = 1 1GtoA: n = 1 Values aremeans ± SEM. CF: Cystic Fibrosis, BMI: Body mass index, FFMI: Fat-freemass index, FEV₁: forced expiratory volume in one second, FMI: fat massindex, FVC: forced vital capacity. The P value represents a comparisonwith Healthy adults. ^(a)P value represents a comparison with CF adultsProtein Digestibility

In healthy young adults, protein digestibility was about 80% in linewith reported rates of spirulina protein (FIG. 3). The average proteindigestibility during feeding and before the pancreatic enzyme intake(t=200-240 min) was significantly lower in the total CF group ascompared to the healthy subjects (p<0.001) which corresponded to 46.5%of the healthy subjects (FIG. 3A). After pancreatic enzyme intakeprotein digestibility increased in CF and reached its maximal value at344 min (=104 min) of 90.3% of the healthy subjects and a plateau indigestibility occurred for approximately 80 minutes.

Stratification of the CF group into adults and children showedcomparable values for protein digestibility during feeding (as % of thehealthy subjects) in both groups (44.7% vs. 48.3%, ns) and a similarkinetic response to pancreatic enzyme intake (FIG. 3B). Between 340 and420 min, protein digestibility reached average (plateau) values of 93.4%vs. 87.4% of the healthy subjects in the CF children vs. CF adults,respectively. No difference was found in average protein digestibilityduring feeding or after pancreatic enzyme intake between CF patientswith and without the homozygous DF 508 gene, or between CF patients withor without nutritional failure. Furthermore, no significant relationshipwas found with lung function in the CF group.

Whole-Body Rate of Appearance and Production Rate of Citrulline

Whole-body citrulline rate of appearance (FIGS. 4A, 4C) and productionrate (FIGS. 4B, 4D) were significantly higher in the CF patients ascompared to the healthy subjects in the postabsorptive state (p<0.05).However, no difference was present between the adults and children withCF. Plasma citrulline concentration was not significantly different inCF as compared to the healthy group (41±4 μmol/L vs. 33±2 μmol/L;p=0.1), Significant higher values were found for whole-body citrullinerate of appearance in the CF patients with nutritional failure ascompared to those without nutritional failure (P<0.05). Whole-bodycitrulline rate of appearance and production rate were not related tolung function or protein digestibility during feeding (data not shown).

Example 3 Fat Digestion Study Materials and Methods

Subjects

The study population consisted of 10 pediatric subjects with CF, ages10-18 years at the time of enrollment and admitted to ArkansasChildren's Hospital for antibiotic treatment of a pulmonaryexacerbation. Furthermore, 9 adults with CF, age of 18 to 35 years atthe time of enrollment were studied. Three of them were admitted toUniversity of Arkansas for Medical Sciences for antibiotic treatment ofa pulmonary exacerbation, and 6 were stable outpatients. The CF subjectswere enrolled with a diagnosis of CF based on universal diagnosticcriteria, abnormal lipid digestion requiring pancreatic enzymereplacement therapy, and improvement in lung function (FEV₁) at the timeof enrollment back to baseline values (determined as FEV₁ in past 12months). Exclusion criteria included established diagnosis of diabetesmellitus, unstable metabolic diseases, and chronic respiratory failurewith cor pulmonale). Written informed consent was obtained and the studywas approved by the Institutional Review Board. This trial is registeredat ClinicalTrials.gov under as NCT01494909.

Composition of the Nutritional Supplement

All subjects received a nutritional supplement, Ensure plus. Thesubjects ingested the nutritional supplement orally according to a sipfeeding protocol (every 20 min) or enterally (when feeding tube ispresent in CF subjects (n=3 pediatric CF)). The dosage of thenutritional supplement for each subject was based on ⅓^(rd) of theirtotal daily protein needs, estimated using Dietary Reference Intakes(DRI). The stable isotopes of [1,1,1-¹³C₃]tripalmitin and[2,2-H₂]palmitic acid were added to measure fat digestion rate (=ratio[1-¹³C]palmitic acid to [2,2-²H₂]palmitic acid). The nutritionalsupplement was mixed with the oral stable isotopes and divided intoequal portions and stored in the refrigerator before use. Pancreaticenzyme (Creon®) intake in CF took place at 2 h into feeding and the dosewas based on the fat content of the meal (4000 u/g fat intake). Nopancreatic enzymes were taken by the healthy adults. Pancreatic enzymeswere obtained from ACH and UAMS pharmacy.

Anthropometric Data and Body Composition

Body weight and height were measured by a digital beam scale andstadiometer, respectively. BMI was calculated by dividing body weight bysquared height. Height, weight and BMI percentiles of the CF subjectswere calculated in accordance with the CF consensus report (1). Wholebody fat mass (FM) and fat-free mass (FFM) were obtained in the patientswith CF by dual-energy X-ray absorptiometry (DXA) (Hologic QDR4500/Version 12.7.3.1 Bedford, Mass.) when the patients were in supineposition. The anthropometric and body composition data were standardizedfor height to obtain BMI, FFMI, and FMI and expressed as percentage ofpublished reference data. Body composition was determined in the wholebody as well as in the arms, legs, and trunk. The DXA procedure was doneonce during hospital stay or the data were copied from subject's filewhen DXA was performed in the preceding month of the study as part of CFcare. Nutritional failure was defined as FFMI<5^(th) percentile inaccordance to previous studies in CF and/or BMI<10^(th) percentile(age≤20 years) or BMI<18 kg/m² (age 21 and older) the Cystic FibrosisFoundation selected BMI cut-off point (3).

Lung Function

Forced expiratory volume in 1 second (FEV₁) and forced vital capacity(FVC) was measured by spirometry (nSpire Health, Longmont, Colo.) in allparticipants and reference equations (2) were used to calculate FEV₁ andFVC % predicted values.

Study Protocol

The study days were performed in the patient's hospital room during thelast days of 2 weeks of antibiotic treatment for a CF exacerbation atArkansas Children's Hospital (CF children) or UAMS (CF adults). Theadult CF patients and the healthy adults were studied at the Centertranslational research at UAMS. The study day started in the earlymorning after overnight fasting and lasted for approx. 8 hours. Bodyweight and height were measured and a catheter was placed in asuperficial vein of the lower arm or the central-venous port for bloodsampling, or a peripheral line already in place for clinical care wasused. After 2 hours in the postabsorptive state, each subject ingestedorally or received enterally (when feeding tube was present in CFsubject) a commercially available nutritional supplement that generallyis used in CF care (Ensure plus) according to a sip feeding protocol(each 20 min) during 6 hours. To the nutritional supplement, the oralisotopes of [1,1,1-¹³C₃]tripalmitin (1 μmol/kg BW/h), and[2,2-H₂]palmitic acid (3 μmol/kg BW/h) were added. After 2 hours offeeding, one serving of pancreatic enzymes (Creon®, 4000 u/g fat intake)was ingested by the CF subjects. No pancreatic enzymes were taken by thehealthy adults. Blood was sampled once for analysis of the naturalenrichment of amino acids. Arterialized-venous blood samples were takenthroughout the study for analysis of concentrations and tracer-traceeratios (TTR) of amino acids.

Biochemical Analysis

Arterialized-venous blood samples were put in Li-heparinized or EDTAtubes (Becton Dickinson Vacutainer system, Franklin Lakes, N.J.) andimmediately put on ice to minimize enzymatic reactions. The blood wascentrifuged and put in 50% sulfosalicyl acid matrices to deproteinizethe plasma, and then instantly frozen and stored at −80° C. untilfurther analyses. All samples obtained were analyzed in a batchaccording to routine measurements (4, 5).

Calculations

[1,1,1-¹³C₃]tripalmitin and [2,2-²H₂]palmitic acid were added (FIG. 1B)to the meal and the ratio between the appearance of free fatty acids fordigestion of triglycerides is measured when 100% of triglycerides aredigested. The ratio between [1-¹³C]palmitic acid/[2,2-²H₂]palmitic acidrepresents the percentage digestion of triglycerides by lipase from thepancreas.

Statistical Analysis

Results are expressed as mean±standard error (SE). The level ofsignificance was set at p<0.05. The statistical package within GraphpadPrism (Version 5.04), and SPSS (version 20) was used for data analysis.

Example 4 Fat Digestion Study Results

The group consisted of 10 children with CF (mean age: 14.9±0.2 y), 9adults with CF (mean age: 28.6±0.8 y), and 8 healthy adults (29.2±1.2 y)(Table 3). Age of the CF adults was not different from the healthyadults. The patients had mild to severe airflow obstruction and werecharacterized on average by reduced values for FFM and FM. FEV₁ and FVCwas significantly lower in the CF adults than in the CF children(P<0.05). Three of the adults and 3 of the children with CF hadnutritional failure based on BMI<10 percentile and/or FFMI<5^(th)percentile.

Compared to healthy subjects, fat digestion rate during feeding (at 240min into the study) were on average 57% and 49% of normal in the wholeCF group (FIG. 5A-5B). Intake of the pancreatic enzymes graduallyincreased fat digestion to 68% of normal at 4 h after the enzyme intake.

Stratification of the CF group in children (n=10, FEV₁:82±1% pred) andadults (n=9, FEV₁:64±3% pred.) showed a lower fat digestion rate (FIG.6A) in CF children during feeding (43 vs. 68% resp., P<0.05) at 240 mininto the study. For the fat digestion rate, there was a significantgroup (P<0.001) and time effect (P<0.01) but no interaction, indicatingthat the increase in the fat digestion rate after pancreatic enzymeintake (FIG. 6B) was comparable in children and adults with CF but theabsolute fat digestion rate remained lower in the CF children even at 4hours after pancreatic enzyme intake (67 vs. 94% resp., P<0.05).

TABLE 3 General characteristics of the children and adults with CF andthe healthy controls Healthy controls CF adults CF children Gender m/f4/4 5/4 6/4 Age y 29.2 ± 1.2 28.6 ± 0.8 14.9 ± 0.2 BMI Kg/m² 23.5 ± 0.421.6 ± 0.5 46.4 ± 3.0 Percentile FFMI % norm 102.3 ± 1.6  90.7 ± 1.592.8 ± 1.1 Nutritional y/n 0/8 3/6 3/7 failure FEV₁ % pred 63.9 ± 3.381.6 ± 1.3 FVC % pred 76.6 ± 3.6 98.0 ± 1.6 CF DF508/DF508: n = 2;DF508/DF508: n = 9; genotype DF508/G542x: n = 2; DF508/1717-1G TODF508/2184delA, A: n = 1 DF508/G1244: n = 1, unknown: n = 2 Values aremeans ± SEM. CF: Cystic Fibrosis, BMI: Body mass index, FFMI: Fat-freemass index, FEV₁: forced expiratory volume in one second, FVC: forcedvital capacityDiscussionProtein Digestion StudyImpaired Protein Digestibility in CF During Feeding and Response toPancreatic Enzyme Intake

In the present study, sip feeds were used as a model that reflectssupplemental enteral feeding by gastrostomy tube as often used toimprove the nutritional status of undernourished CF patients. Tubefeeding is administered mostly at night at continuous infusion over 6 to12 hours and pancreatic enzymes are given at the start of the feeding.The observed protein digestibility during feeding of 47% of normal in CFindicates a severe reduction of protein digestibility that wascomparable in adults and children with CF. The dose of pancreaticenzymes was determined on an individual basis taking the fat content andrate of administration of the sip feeding into consideration. It wasobserved that 100 min were needed after intake of the pancreatic enzymesbefore protein digestibility reached its maximal value of 90% of normalin CF. A comparable response in protein digestibility after pancreaticenzyme intake was present in children and adults with CF.

Approaches to Circumvent the Impaired Protein Digestibility in CF

There are several ways to circumvent the reduced protein digestibilityin CF patients while they are on pancreatic enzyme replacement therapy,i.e., adjusting the type of protein intake into more slowly digestedproteins, intake of hydrolyzed proteins or free dietary amino acids,changing the timing of pancreatic enzyme intake, and/or by modifying thedose/composition (lipase, protease, amylase) of the pancreatic enzymecapsules. These results indicate that when meals are consumed consistingof proteins that are slowly digested (e.g., casein protein), whichmimics the sip feeding protocol used herein, it takes nearly 2 hours forthe enzyme activity to achieve a maximal effect in CF patients.

The normalization of protein digestibility that occurred afterpancreatic enzyme intake in the studied CF patients suggests that theamount of protease in the pancreatic enzymes is sufficient. However,normalization of protein digestibility was severely delayed as it tooknearly 2 hours before protein digestibility was normalized, suggestingthat the pancreatic enzyme capsules need to be modified duringcontinuous feeding to make the enzymes more quickly available andactive. Pancreatic enzyme products are labeled according to the amountof lipase they contain. All products also contain protease and amylase,but the labeled and actual amounts of these two enzymes may differ fromproduct to product even when labeled lipase amounts are the same. Thedata suggests that the pancreatic enzyme products need to work fasterduring meal intake and that the labeling of the products should alsocontain the protease activity. For pancreatic enzymes to be effectiveproteases, it is crucial that they are available when protein in thefood reaches the proximal small intestine, which is the place whereamino acid uptake mainly takes place. These results indicate that a fastresponse is essential for the proteases, and therefore the capsules needto be opened to release the proteases in the last part of the duodenumwhen pH is less acidic, while for the lipases a much slower response isneeded as they are irreversibly inactivated at pH 4.0 or lower. The doseand timing of administered pancreatic enzymes in CF should therefore bebased on multiple factors like the composition of the diet (amountfat/protein) in order to improve both fat and protein digestibilitysimultaneously and on factors like gastric acid secretion, pH atdifferent levels of the small and large intestine, patterns of gastricemptying, and bile acid composition and concentration.

Whole-Body Citrulline Production as Measure of Gut Function

In the present study, plasma citrulline concentration was elevated inthe CF as compared to the healthy subjects. Therefore, both in CFpatients at the end of hospitalization for an acute exacerbation as instable CF outpatients, gut mucosal function seem to be unaffected.Citrulline is an amino acid released almost exclusively from the smallbowel enterocyte mucosal mass as it is not a protein component and assuch not incorporated in enteral food or endogenous proteins. In thepresent study, whole-body citrulline rate of appearance and productionrate were also elevated in CF but not related to protein digestion rateduring feeding or with the average protein digestion rate obtained afterpancreatic enzyme intake in CF. No association was found betweenwhole-body citrulline rate of appearance or production and lung functionor age in CF.

However, to measure overall digestibility of meals with proteins, someor all proteins should be labeled with ¹⁵N. Isotope ratio measurementsare necessary to be able to calculate the protein digestion rate andthese analytical techniques can be easily implemented on available GC-MSor LC-MS machines in the clinic. As the stable isotope-based methodmeasures protein digestibility, no information on amino acid absorptionwas obtained. As the elevated whole-body citrulline productionindirectly indicates that mucosa function is normal in CF, a directmeasurement of amino acid absorption could be made by using marker aminoacids that are not metabolized in the body (e.g., labeled amino acidanalogues), assuming that the marker absorption rates are representativefor the amino acid absorption rate in general. Furthermore, in order totest this method, CF patients were studied during continuous (sip)feeding as it closely reflects the situation present in patients with agastrostomy tube during overnight feeding. In conclusion, proteindigestibility during continuous feeding as measured by this novel andeasy-to-use stable isotope technique is severely compromised in patientswith CF and normalization is possible but delayed after pancreaticenzyme intake. Gut (mucosa) function as measured by whole-bodycitrulline production is not affected in CF.

Fat Digestion Study

Maldigestion of fat is likely if an individual ingesting 100 g of fatper day excretes more than 7 g of fat in a 24-hour period. In thepresent study sip feeds were used to reflect supplemental enteralfeeding by gastrostomy tube (GT) as this is often used to improve thenutritional status of undernourished CF patients. GT feeding isadministered mostly at night at continuous infusion over 6-12 hours. Thedose of PERT is worked out on an individual basis taking the type, fatcontent, and rate of administration of the feeding into consideration.In practice, a starting dose of one to two capsules of the patient'susual enzyme preparation is given at the start and at the end of thefeeding. The dose is then titrated against symptoms rather than the fatcontent of the feed. If bolus feeds are being given or the feeding isbeing infused over a short period of time, larger enzyme doses may berequired due to the faster rate of fat infusion.

The following references are relied on herein.

-   1. Borowitz D et al. J Pediatr Gastroenterol Nutr 2002; 35:246-59.-   2. Wolfe et al., Isotope Tracers in Metabolic Research: Principles    and Practice of Kinetic Analysis. New York: Wiley, New York, 2005.-   3. Mille C E et al. Clin Chest Med 2007; 28:319-30.-   4. Andersen D H et al. Am. J. Dis. Child. 1945; 69:221.-   5. Lavik P S et al. Pediatrics 1952; 10:667-76.

What is claimed is:
 1. A method for determining reduced fatdigestibility in an individual in need of such determination, comprisingthe steps of: orally administering a meal comprising a [¹³C]-labeledtriglyceride and a [²H]-labeled free fatty acid tracer to saidindividual and a control subject; and measuring the amount of[¹³C]-labeled fatty acid formed after digestion of said [¹³C]-labeledtriglyceride and measuring the amount of [²H]-labeled free fatty acidtracer in a biological sample of said individual and said controlsubject to determine a ratio of [¹³C]-labeled fatty acid to [²H]-labeledfree fatty acid tracer for said individual and said control subject;wherein a lower ratio of [¹³C]-labeled fatty acid to [²H]-labeled freefatty acid tracer in said individual compared to the ratio of[¹³C]-labeled fatty acid to [²H]-labeled free fatty acid tracer fromsaid control subject indicates that said individual has a reducedcapacity to digest fat.
 2. The method of claim 1, wherein said[²H]-labeled free fatty acid tracer is [2,2-²H₂]-palmitic acid,[2,2-²H₂]-oleic acid, or (7,7,8,8-D₄)palmitic acid.
 3. The method ofclaim 1, wherein said [¹³C]-labeled triglyceride is[1,1,1-¹³C₃]-tripalmitin, [1,1,1-¹³C₃]-triolein, [U-¹³C₁₆]-tripalmitin,or [U-¹³C₁₈]-triolein.
 4. The method of claim 1, wherein said[¹³C]-labeled fatty acid formed after digestion of said [¹³C]-labeledtriglyceride is [1-¹³C]-palmitic acid, [1-¹³C]-oleic acid,[U-¹³C₁₆]-palmitic acid, or [U-¹³C₁₈]-oleic acid.
 5. The method of claim1, wherein said individual has cystic fibrosis.
 6. The method of claim1, wherein said biological sample is blood or plasma.
 7. A method forobtaining fat digestibility profile in an individual, comprising thesteps of: orally administering a meal comprising a [¹³C]-labeledtriglyceride and a [²H]-labeled free fatty acid tracer to saidindividual; measuring the amount of [¹³C]-labeled fatty acid formedafter digestion of said [¹³C]-labeled triglyceride and measuring theamount of [²H]-labeled free fatty acid tracer in a biological sample ofsaid individual to determine a ratio of [¹³C]-labeled fatty acid to[²H]-labeled free fatty acid tracer; and repeating said measuring stepone or more times to provide a fat digestibility profile for saidindividual.
 8. The method of claim 7, wherein said [²H]-labeled freefatty acid tracer is [2,2-²H₂]-palmitic acid, [2,2-²H₂]-oleic acid, or(7,7,8,8-D₄)palmitic acid.
 9. The method of claim 7, wherein said[¹³C]-labeled triglyceride is [1,1,1-¹³C₃]-tripalmitin,[1,1,1-¹³C₃]-triolein, [U-¹³C₁₆]-tripalmitin, or [U-¹³C₁₈]-triolein.